Project Summary The burden of snakebite is borne by low- to middle-income countries, affecting young adults and family providers living in rural communities with limited access to public healthcare. Five million people are bitten by snakes every year, and of those more than 100,000 die and approximately 400,000 are left severely disfigured or without the affected limbs. While antivenom can effectively reverse the toxic effects of envenomation, the methods for producing antivenom are archaic and involve immunization and subsequent plasmapheresis of dedicated antivenom producing horses and sheep. As a result, antivenom suffers from batch-to-batch variation, is unaffordable by those most affected, and is comprised predominantly of non-therapeutic, highly immunogenic animal-derived pollutants that severely complicate treatment. In addition, antivenom is toxin-specific and thus only effective against the venom to which it was raised. Since most snakebite victims cannot identify the envenoming animal, polyclonal antivenom is made by mixing together antibodies from horses immunized with multiple medically important snake species. Currently, no single cross-reactive, recombinantly produced therapeutics are available for snakebite treatment, but there exists an abundance of cross-toxin binding proteins that are both non-immunogenic and amenable to rational redesign. We will use recombinant expression technologies and protein x-ray crystallography to identify key elements that are conserved between snake toxins from multiple species, and thus susceptible to broad neutralization by these proteins. Through the comprehensive characterization of antitoxin binding at the atomic level, we will gain an understanding how these details might be exploited by structure-guided protein redesign. Lastly, we will use this information to optimize the expression, breadth, potency, stability, and rationally assembly of next- generation antivenoms with the potential to treat snakebite in multiple developing nations.